Commitment to the T and natural killer T (NKT) cell lineages is determined during alphabeta T cell receptor (TCR)-mediated interactions of common precursors with ligand-expressing cells in the thymus. Whereas mainstream thymocyte precursors recognize major histocompatibility complex (MHC) ligands expressed by stromal cells, NKT cell precursors interact with CD1d ligands expressed by cortical thymocytes. Here, we demonstrated that such homotypic T-T interactions generated "second signals" mediated by the cooperative engagement of the homophilic receptors Slamf1 (SLAM) and Slamf6 (Ly108) and the downstream recruitment of the adaptor SLAM-associated protein (SAP) and the Src kinase Fyn, which are essential for the lineage expansion and differentiation of the NKT cell lineage. These receptor interactions were required during TCR engagement and therefore only occurred when selecting ligands were presented by thymocytes rather than epithelial cells, which do not express Slamf6 or Slamf1. Thus, the topography of NKT cell ligand recognition determines the availability of a cosignaling pathway that is essential for NKT cell lineage development.

SummaryCharacterization of single antibody lineages within infected individuals has provided insights into the development of Env-specific antibodies. However, a systems-level understanding of the humoral response against HIV-1 is limited. Here, we interrogated the antibody repertoires of multiple HIV-infected donors from an infection-naive state through acute and chronic infection using next-generation sequencing. This analysis revealed the existence of “public” antibody clonotypes that were shared among multiple HIV-infected individuals. The HIV-1 reactivity for representative antibodies from an identified public clonotype shared by three donors was confirmed. Furthermore, a meta-analysis of publicly available antibody repertoire sequencing datasets revealed antibodies with high sequence identity to known HIV-reactive antibodies, even in repertoires that were reported to be HIV naive. The discovery of public antibody clonotypes in HIV-infected individuals represents an avenue of significant potential for better understanding antibody responses to HIV-1 infection, as well as for clonotype-specific vaccine development.

Signal transduction initiated by B cell Ag receptor (BCR) cross-linking plays an important role in the development and activation of B cells. Therefore, considerable effort has gone into determining the biochemical signaling events initiated by the BCR and delineating which events participate in specific biological responses to Ag. We used two inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 1 and MEK2, PD98059, and U0126, to assess the role the Ras-mitogen-activated protein kinase pathway plays in several BCR-induced responses. PD98059 or U0126 treatment substantially inhibited the BCR-induced activation of the extracellular signal-regulated kinase (ERK) forms of mitogen-activated protein kinase in the immature B cell line WEHI-231, in immature splenic B cells, and in mature splenic B cells. However, MEK-ERK inhibition did not block BCR-induced growth arrest or apoptosis of WEHI-231 cells or apoptosis of immature splenic B cells, indicating that the MEK-ERK pathway is not required for these events. In contrast, PD98059 and U0126 treatment did inhibit the up-regulation of specific BCR-induced proteins, including the transcription factor Egr-1 in WEHI-231 and mature splenic B cells, and the CD44 adhesion molecule and CD69 activation marker in mature splenic B cells. Moreover, both inhibitors suppressed BCR-induced proliferation of mature splenic B cells, in the absence and in the presence of IL-4. Therefore, activation of the MEK-ERK pathway is necessary for a subset of B cell responses to Ag.

We have previously developed a combination therapy (CT) using anti-CD3 monoclonal antibodies together with islet-(auto)antigen immunizations that can more efficiently reverse type 1 diabetes (T1D) than either entity alone. However, clinical translation of antigen-specific therapies in general is hampered by the lack of biomarkers that could be used to optimize the modalities of antigen delivery and to predict responders from nonresponders. To support the rapid identification of candidate biomarkers, we systematically evaluated multiple variables in a mathematical disease model. The in silico predictions were validated by subsequent laboratory data in NOD mice with T1D that received anti-CD3/oral insulin CT. Our study shows that higher anti-insulin autoantibody levels at diagnosis can distinguish responders and nonresponders among recipients of CT exquisitely well. In addition, early posttreatment changes in proinflammatory cytokines were indicative of long-term remission. Coadministration of oral insulin improved and prolonged the therapeutic efficacy of anti-CD3 therapy, and long-term protection was achieved by maintaining elevated insulin-specific regulatory T cell numbers that efficiently lowered diabetogenic effector memory T cells. Our validation of preexisting autoantibodies as biomarkers to distinguish future responders from nonresponders among recipients of oral insulin provides a compelling and mechanistic rationale to more rapidly translate anti-CD3/oral insulin CT for human T1D.

The Src family kinase Fyn is expressed in T cells and has been shown to phosphorylate proteins involved in TCR signaling, cytoskeletal reorganization, and IL-4 production. Fyn-deficient mice have greatly decreased numbers of NKT cells and have thymocytes and T cells with compromised responses following Ab crosslinking of their TCRs. Herein we have addressed the role of Fyn in peptide/MHC class II-induced CD4+ T cell responses. In Fyn-deficient mice, CD4+ T cells expressing the DO11.10 TCR transgene developed normally, and the number and phenotype of naive and regulatory DO11.10+CD4+ T cells in the periphery were comparable with their wild-type counterparts. Conjugation with chicken OVA peptide 323–339-loaded APCs, and the subsequent proliferation in vitro or in vivo of DO11.10+ Fyn-deficient CD4+ T cells, was virtually indistinguishable from the response of DO11.10+ wild-type CD4+ T cells. Proliferation of Fyn-deficient T cells was not more dependent on costimulation through CD28. Additionally, we have found that differentiation, in vitro or in vivo, of transgenic CD4+ Fyn-deficient T cells into IL-4-secreting effector cells was unimpaired, and under certain conditions DO11.10+ Fyn-deficient CD4+ T cells were more potent cytokine-producing cells than DO11.10+ wild-type CD4+ T cells. These data demonstrate that ablation of Fyn expression does not alter most Ag-driven CD4+ T cell responses, with the exception of cytokine production, which under some circumstances is enhanced in Fyn-deficient CD4+ T cells.

Mutations in a number of signaling components in mice can lead to strong autoimmune phenotypes. In some cases, these mutations likely compromise important feedback inhibitory pathways that downregulate antigen receptor signaling. For example, a deficiency of Lyn leads to a severe lupus-like autoimmunity. This autoimmunity may result from loss of a feedback inhibitory pathway in which Lyn phosphorylates CD22, triggering recruitment of the tyrosine phosphatase SHP-1 to the plasma membrane, which then dampens BCR signaling. Loss of Lyn also compromises an inhibitory pathway involving Fc gamma RIIb and SHIP, an inositol phosphatase. Mutation of Fyn exacerbates the autoimmunity caused by loss of Lyn. This may be due in part to a nonimmunological compromise in the integrity of the podocytes in the kidney, which may make the kidneys more susceptible to immune complex-induced damage. Fyn-deficient mice exhibit a number of immunological abnormalities and also exhibit some autoimmunity, although this is less severe than what is seen in Lyn-deficient mice. Recently a gain of function mutation in CD45 that may enhance activity of Src family tyrosine kinases has also been found to cause autoimmune disease, suggesting that the level of Src family tyrosine kinase activity is an important determinant of immune tolerance. Finally, several studies suggest that there is a significant interaction between Src family tyrosine kinases and the Fas pathway that is important for self-tolerance. Although these studies are still at an early stage, it seems clear that alterations in regulators of antigen receptor signaling can contribute to autoimmunity.

Immunologists have developed a range of in vitro techniques for probing the receptor mediated response of cells comprising the immune system. An important and ubiquitous method is the use of antibodies in either soluble or aggregated form to engage cell surface receptors and transmit a signal. Models of cell and molecular interactions, derived from the use of these antibodies, form the basis of our efforts to understand and explain the corresponding in vivo systems. However, interpreting in vitro experiments and distinguishing between alternative models is difficult. This complexity is illustrated here using B cell stimulation by surface immunoglobulin and CD40. The fluorescent cell labelling dye carboxyfluorescein, diacetate, succinimidyl ester (CFSE) is used to show that many anti-Ig and CD40 stimulatory agents, used to assess the role of B cells and lymphokines, are partial agonists. By modelling each step in B cell signalling, activation and division it is possible to show that small changes in signal contributed by a second receptor can generate numerous distinct dose response curves that are highly dependent on the "efficacy" of signal transmission by the primary ligand and the number of cell divisions taken in culture. Differences in dose response curves become particularly striking if the primary activating stimulus is a partial agonist. Although exemplified here with B cell stimulation the conclusions are applicable to other in vitro activation systems and suggest ways to improve both the design and interpretation of in vitro experiments.*Corresponding author. Tel.: 61 2 9565 6121. Fax: 61 2 9565 5103.

«Summary Early theories of antibody production by B cells achieved considerable success in predicting B cell behaviour with simple deductive models. One such model, the one signal theory, postulated that the antigen receptor on B cells played only a passive non-signalling role in focusing non-specific activating signals to the B cell surface. This prediction is at least partially consistent with recent discoveries concerning the helper signals delivered to B cells by T cells. Here, we re-examine the foundation of this theoretical prediction with the benefit of recent information. The experimental basis for the theory was a study of B cell activation by LPS and, in particular, the interpretation of a bell-shaped dose-response curve. The logic applied is appropriate to explain some, but not all, forms of B cell behaviour because, as is now clear, the role played by tbe antigen signal varies depending upon the method of activation. This re-examination suggests an alternative interpretation of the LPS-induced bell-shaped curves that incorporates a role for an antigen signal. If correct, the mechanism would ensure that T-independent responses are drawn from low affinity precursors.